This U19 Cooperative Agreement grant application is designed to perform studies of preclinical optimization of an antimicrobial peptide, retrocyclin in combination with cyanovirin-N-secreting lactobacilli (CV-N/LAB) that will have potent anti-HIV activity against cell-free and cell-associated virus, be non-toxic and noninflammatory to the cervical and vaginal tissues and in monkeys. An optimal combination of topical microbicide agents with these properties will be chosen based on a highly interactive approach between four projects and three cores. Together, we will employ an array of in vitro and ex vivo laboratory tests of efficacy and toxicity, in vivo safety tests in monkey models, and studies to optimize the formulation of our compounds. Our overall hypothesis is that a combination of retrocyclin and CV-N /LAB, two physiologically relevant compounds, will have high anti-HIV activity and low toxicity under in vitro and in vivo conditions. The aims of this grant application are: 1) Optimization of a Lactobacillus bacteria secreting cyanovirin microbicide (Project 1, Ramaratnam), 2) Development of a retrocyclin microbicide (Project 2, Cole), 3) Testing lead candidate microbicides for antiviral activity and toxicity in vaginal organ culture (Project 3, Gupta), 4) Evaluation of microbicide safety and toxicity in pigtail macaques (Project 4, Patton). The scientific studies in these four projects will be supported by the Administrative Core (Core A, Gupta), Formulation Core (Core B, Rohan) and cell-cell transmission assay facility (Core C, Southern Research Institute). In designing the scope of the proposed topical microbicide program, we have focused on extensively evaluating safety and toxicity of candidate microbicides utilizing in vitro studies, tissue explants and animal models, as well as an expanded and innovative organ culture model to study the interaction of HIV-1 and common STI in evaluating antiviral activity of microbicides in the presence of STI. The development of a combination microbicide product that incorporates CV-N/LAB and retrocyclin would provide a novel strategy which is expected to have potent anti-HIV activity without inducing cellular toxicity.